Toxicity of drug candidates accounts for a significant portion of attrition during exploratory development. Following development and commercialization, therapeutic agents exhibiting adverse reactions are less competitive and may ultimately fail in the market place. Implementing a Discovery screen to weed out compounds with potential toxicity early would enable all Discovery candidates entering development to achieve a higher survival rate. Following development, the selected drugs would be more likely to enjoy a competitive advantage and a more favorable therapeutic index.
One common mode of toxicity is the formation of electrophilic reactive metabolites, which manifest their toxicity by covalent binding to nucleophilic groups present in vital cellular proteins and nucleic acids (1-2). Although not all toxicological manifestations are attributable to reactive metabolites, a significant body of literature suggests that inadequate detoxification of chemically reactive metabolites formed as a result of drug bioactivation is a pathogenic mechanism for tissue necrosis (3-4), carcinogenicity (5), teratogenicity (6) and immune mediated toxicity (7).
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This invention provides a method for identifying drug candidates which produce reactive metabolites which comprises:
(a) incubating said drug candidates with a microsomal drug metabolizing enzyme system in the presence of glutathione and;
(b) detecting glutathione conjugates formed in step (a).
This invention further provides a method for identifying drug candidates which produce reactive metabolites which is a high throughput method.
This invention further provides a method for identifying drug candidates which produce reactive metabolites wherein said drug metabolizing enzyme system is a human liver system.
This invention also provides a method for identifying drug candidates which produce reactive metabolites which comprises: incubating said drug candidates with a microsomal drug metabolizing enzyme system in the presence of glutathione and detecting glutathione conjugates by tandem mass spectometry.
This invention provides a high throughput method of identifying candidates with the potential to produce reactive metabolites. It exploits one of the natural mechanisms for eliminating reactive intermediates: conjugation with glutathione. Through its nucleophilic sulfhydryl group, glutathione protects vital cellular constituents against chemically reactive species which bind to the reactive electrophilic moiety, to form stable S-substituted adducts (8). The detoxification of the acetaminophen electrophilic metabolite by glutathione is one of the classic examples of that protection.
A universal analytical method for detecting glutathione conjugates, which are generated by in vitro incubation with human liver microsomal drug metabolizing enzyme systems and with glutathione, will identify compounds that undergo bioactiviation to reactive metabolites. This analytical method utilizes tandem mass spectrometry. During collision induced dissociation, all glutathione adducts undergo the neutral loss of the pyroglutamic acid moiety (129 Da). The diagnostic neutral loss of 129 Da allows the specific detection of any glutathione conjugates that have been generated via metabolic activation followed by conjugation (9).
The method of this invention is reliable, rapid, simple and amenable to high throughput automation. The incubation step can easily be automated following the heated block 96 well incubation method developed by J. Janiszewski. The sample preparation and extraction step is automated using Quadra 96 SPE (10). The analytical step is particularly simple requiring no compound specific optimization. Although we have utilized chromatography with 7-minute runtime, the specificity of the assay suggests that a runtime of 1 minute per sample is feasible. Consequently, throughput of more than 1000 compounds a day can be attained by utilizing the dual column switching system.
The method will detect most toxic compounds whose toxicity is mediated by reactive metabolites generated by bioactivation with drug metabolizing oxidative P450 systems. It will not detect reactive metabolites that do not form stable adducts with glutathione (such as free radicals) or those formed by non-microsomal enzymes.